Journal: bioRxiv

Article Title: Discovery of novel compounds and target mechanisms using a high throughput, multiparametric phenotypic screen in a human neuronal model of Tuberous Sclerosis

doi: 10.1101/2024.02.22.581652

Figure Lengend Snippet: (A) mTOR pathway: TSC1/TSC2 proteins negatively regulate the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling, a major regulator of cell growth and proliferation. (B) CRISPR/Cas9 was used on two control ( TSC2 +/+ ) iPS cell lines (11a and 20b) to generate a collection of TSC2 +/+ , TSC2 −/− and TSC2 +/− iPS cell clones. (C) Predicted TSC2 genotype based on PCR Sanger Sequencing and TSC2 protein levels were confirmed using immunoblotting assays. (D) Cortical excitatory neurons (“NGN2” neurons) were produced from the different CRISPR/Cas9-edited isogenic TSC2 clones using a transcriptional programming approach driven by overexpression of the proneuronal transcription factor NEUROG2 (NGN2). ( E ) High content imaging characterization of TSC2 +/+ vs TSC2 −/− NGN2 neurons showed an increase in cell soma size and upregulation of pS6 kinase in TSC2 −/− neurons, which could be reversed by chronic Rapamycin treatment, validating the disruption of mTOR signaling in these cells. (F) Immunocytochemistry of TSC2 +/+ and TSC2 −/− neurons (detected with MAP2 antibody) showing upregulation of the mTOR effector pS6 in TSC2−/− neurons, which can be corrected with Rapamycin treatment. ( G ) Quantification of morphometric (cell size) phenotype and rescue with mTOR modulator Rapamycin

Article Snippet: Primary immunoreagents used in this study included antibodies detecting the cytoskeletal pan-neuronal markers MAP2 (microtubule associated protein 2) [Abcam; ab5392 (1:3,000) or Novus Biological; NB300-213 (1:3000)] and β-III TUBULIN [TUJ1, BioLegend; #801202 (1:1,000)], a human nuclear-specific antigen [(EMD Millipore; MAB1281 (1:1000)], as well as the mTOR effector phosphor S6 ribosomal protein (pS6 kinase) [Cell Signaling; #4858 (1:1000)].

Techniques: CRISPR, Control, Clone Assay, Sequencing, Western Blot, Produced, Over Expression, Imaging, Disruption, Immunocytochemistry

Journal: bioRxiv

Article Title: Discovery of novel compounds and target mechanisms using a high throughput, multiparametric phenotypic screen in a human neuronal model of Tuberous Sclerosis

doi: 10.1101/2024.02.22.581652

Figure Lengend Snippet: Chronic mTOR inhibition reverses functional excitability phenotype in TSC2 −/− human neurons. (A) Assay timeline for cell culture, compound interventions (treatment regimens A1, B1 and C1), and optical physiology measurements. (B-C) Recovery of functional phenotype was tested using mTOR inhibitors at different doses and treatment regimens, revealing that long chronic dosing (Regimen A1) is necessary to see broad recovery of the functional phenotype, though partial recovery of some features is seen with shorter dosing regimens. (B) Functional phenotype rescue was quantified using a normalized LDA disease score, with assay wells for each condition normalized so that TSC2-KO-112 vehicle controls have a mean value of 0 while TSC2-WT-3 vehicle controls have a mean value of 1. (C) Magnitude and diversity of phenotypic rescue for each treatment regimen was plotted using a similar effect size heatmap used in , with the first column representing phenotypic differences between vehicle controls and remaining columns showing effect of three different mTOR modulators (at two different concentrations) on TSC2-KO-112 neurons. Experimental conditions showing a color opposite (in the Cohen’s D scale) to the phenotypic difference indicate functional phenotype reversal.

Article Snippet: Primary immunoreagents used in this study included antibodies detecting the cytoskeletal pan-neuronal markers MAP2 (microtubule associated protein 2) [Abcam; ab5392 (1:3,000) or Novus Biological; NB300-213 (1:3000)] and β-III TUBULIN [TUJ1, BioLegend; #801202 (1:1,000)], a human nuclear-specific antigen [(EMD Millipore; MAB1281 (1:1000)], as well as the mTOR effector phosphor S6 ribosomal protein (pS6 kinase) [Cell Signaling; #4858 (1:1000)].

Techniques: Inhibition, Functional Assay, Cell Culture

Journal: bioRxiv

Article Title: Discovery of novel compounds and target mechanisms using a high throughput, multiparametric phenotypic screen in a human neuronal model of Tuberous Sclerosis

doi: 10.1101/2024.02.22.581652

Figure Lengend Snippet: ( A ) Timeline of two RNA sequencing experiments profiling TSC2 −/− (TSC2-KO-112 or “KO”) and TSC2 +/+ (TSC2-WT-3 or “WT”) iPS cell-derived NGN2 neurons co-cultured with rodent glial cells. In Experiment 2, neurons were treated with 0.1% DMSO, Everolimus (“Evero”, 2 nM or 1000 nM), Rapamycin (“Rapa”, 2 nM or 1000 nM), or Torin (7 nM or 300 nM) using one of three treatment regimens. ( B ) UpSet plot of genes that are significantly differentially expressed between untreated (Exp. 1) or 0.1% DMSO-treated (Exp. 2) TSC2 KO neurons and TSC2 WT neurons. 7,080 “high-confidence” genes (red and blue bars) are shared. ( C ) Most frequent reactome pathway types enriched in high confidence differentially expressed genes. Top-level terms with ≥10 significantly enriched pathways are shown. ( D ) Neuronal systems reactome pathways significantly enriched in high-confidence down-regulated genes. ( E ) Volcano plot of 7,080 high-confidence genes in Exp. 2. Members of the “Protein-protein interactions at synapses” reactome pathway [R-HSA-6794362] are highlighted in red or blue. ( F ) Effect of drug treatment on high-confidence genes in Exp. 2. with genes sorted by the effect of TSC2 knockout in 0.1% DMSO-treated neurons (far left column). All other columns compare drug-treated TSC2 KO neurons to 0.1% DMSO-treated TSC2 KO neurons in that treatment regimen. Log 2 fold-change values >2 or <-2 have been flattened to 2 or −2. ( G ) Percentage of the 7,080 high-confidence genes whose expression is significantly reversed by compound treatment in TSC2 KO neurons across compound and treatment regimens. ( H ) Overlap between genes that are significantly reversed by high dose regimen A2 treatments of Everolimus, Rapamycin, and Torin in TSC2 KO neurons. ( I ) Effects of TSC2 knockout (far left column) or high dose regimen A2 treatments of mTOR modulators on TSC2 KO expression of high-confidence genes encoding ion channels. Log 2 fold-change values >2 or <-2 have been flattened to 2 or −2. ( J ) Effects of high dose regimen A2 treatments on high-confidence genes in the “Protein-protein interactions at synapses” reactome pathway [R-HSA-6794362]. Log 2 fold-change values >2 or <-2 have been flattened to 2 or −2.

Article Snippet: Primary immunoreagents used in this study included antibodies detecting the cytoskeletal pan-neuronal markers MAP2 (microtubule associated protein 2) [Abcam; ab5392 (1:3,000) or Novus Biological; NB300-213 (1:3000)] and β-III TUBULIN [TUJ1, BioLegend; #801202 (1:1,000)], a human nuclear-specific antigen [(EMD Millipore; MAB1281 (1:1000)], as well as the mTOR effector phosphor S6 ribosomal protein (pS6 kinase) [Cell Signaling; #4858 (1:1000)].

Techniques: RNA Sequencing, Derivative Assay, Cell Culture, Protein-Protein interactions, Knock-Out, Expressing

Journal: bioRxiv

Article Title: Discovery of novel compounds and target mechanisms using a high throughput, multiparametric phenotypic screen in a human neuronal model of Tuberous Sclerosis

doi: 10.1101/2024.02.22.581652

Figure Lengend Snippet: (A) Assay timeline: Excitatory NGN2 neurons from 20b-TSC2-WT-3 ( TSC2 WT) and 20b-TSC2-KO-112 ( TSC2 KO) iPS cell lines were were plated (DIV0, day in vitro 0) and co-cultured with rodent primary astrocytes and synaptic phenotype and pharmacological rescue were assessed on DIV45 using optical physiology measurements. Neurons were treated with either 2 nM or 10 µM Everolimus using a chronic and multi-intervention treatment regime (treatment at DIV24, DIV39, and acutely at DIV45 ∼1hr prior (and during) functional imaging. (B) Schematic of synaptic imaging protocol. To assess synaptic function, a Cre-based recombination approach was used to allow for orthogonal expression of the two optical physiology components, resulting in two distinct neuronal populations: those expressing the channelrhodopsin CheRiff, and those expressing the voltage reporter QuasAr. Three pulses of blue light were delivered to the neurons (as depicted), resulting in opening of the channelrhodopsin, depolarization of the membrane of CheRiff-expressing neurons, and subsequent action potential firing and synaptic transmitter release. The presence of QuasAr in the remaining neurons allowed for detection of changes in membrane voltage resulting from postsynaptic potentials due to neurotransmitter binding to glutamate receptors (GABAzine was included in imaging buffer to block inhibitory signaling). (C) Excitatory postsynaptic potential waveforms (expressed as changes to the fluorescence of the voltage reporter QuasAr) under vehicle (0.1% DMSO) or Everolimus conditions for TSC2 KO vs. TSC2 WT neurons. TSC2 KO neurons showed a dramatically reduced postsynaptic potential that is partially rescued with increasing concentrations of the mTOR modulator Everolimus. 24 culture wells were measured per condition and n = the number of individual neurons in which a postsynaptic potential was detected. Note that many fewer TSC2 KO neurons showed postsynaptic potentials as compared to TSC2 WT despite an equal number of samples across conditions.

Article Snippet: Primary immunoreagents used in this study included antibodies detecting the cytoskeletal pan-neuronal markers MAP2 (microtubule associated protein 2) [Abcam; ab5392 (1:3,000) or Novus Biological; NB300-213 (1:3000)] and β-III TUBULIN [TUJ1, BioLegend; #801202 (1:1,000)], a human nuclear-specific antigen [(EMD Millipore; MAB1281 (1:1000)], as well as the mTOR effector phosphor S6 ribosomal protein (pS6 kinase) [Cell Signaling; #4858 (1:1000)].

Techniques: In Vitro, Cell Culture, Functional Assay, Imaging, Expressing, Membrane, Binding Assay, Blocking Assay, Fluorescence

Journal: bioRxiv

Article Title: Discovery of novel compounds and target mechanisms using a high throughput, multiparametric phenotypic screen in a human neuronal model of Tuberous Sclerosis

doi: 10.1101/2024.02.22.581652

Figure Lengend Snippet: (A) Normalized pS6 expression in TSC2 +/+ neurons treated with DMSO (n= 464 replicate wells) and TSC2 −/− neurons treated with DMSO (n= 432 wells), 1 μM hit compound (n=1246 wells, 2 replicates per compound for 623 hit compounds), 5 μM hit compound (n=1241 wells, generally 2 replicates per compound), 0.3 μM Torin-1 (n=84 wells), or 30 μM XE-991 (n=16 wells). (B) Number of 210 top hit compounds active (LDA>0.3) at 3.33 μM in a chronic (DIV10+24+31) or acute (30-90min) treatment regimen. (C) Overlap between active groups in B. ( D) Example concentration-response curves for two mTOR modulators (Everolimus and Torin-1) and two top hit compounds (QS-035 and QS-491). (E) Principal component analysis of functional behavior in a chronic treatment regimen for TSC2 KO neurons treated with 0.3 μM Torin-1 or 30 μM XE-991 (left panel) or 3.33 μM library compound colored by activity category (right panel). DMSO-treated TSC2 KO and TSC2 WT controls are shown in both panels. ( F) Principal component analysis of functional behavior in a chronic treatment regimen for TSC2 KO neurons treated with potassium channel blockers or activators vs. DMSO-treated KO and WT controls.

Article Snippet: Primary immunoreagents used in this study included antibodies detecting the cytoskeletal pan-neuronal markers MAP2 (microtubule associated protein 2) [Abcam; ab5392 (1:3,000) or Novus Biological; NB300-213 (1:3000)] and β-III TUBULIN [TUJ1, BioLegend; #801202 (1:1,000)], a human nuclear-specific antigen [(EMD Millipore; MAB1281 (1:1000)], as well as the mTOR effector phosphor S6 ribosomal protein (pS6 kinase) [Cell Signaling; #4858 (1:1000)].

Techniques: Expressing, Concentration Assay, Functional Assay, Activity Assay

Journal: BMC Cancer

Article Title: Radical nephrectomy and regional lymph node dissection for locally advanced type 2 papillary renal cell carcinoma in an at-risk individual from a family with hereditary leiomyomatosis and renal cell cancer: a case report

doi: 10.1186/s12885-016-2272-7

Figure Lengend Snippet: Western blotting. Western blotting for surgically resected tissues (M: marker, N: normal tissue, T1-3: three different parts of tumor tissues). In a patient with cT3bN1M1 clear cell renal cell carcinoma (ccRCC-1) who received preoperative axitinib as well as the current patient, tumor tissues obtained by nephrectomy after axitinib treatment showed heterogeneous changes. Some tumor tissue (T2 and T3) showed much lower expression of pAkt (Ser-473), pAkt (Thr-308), and pS6 than other tissue (T1). Similarly, in the other patients with cT any N1M any ccRCCs (ccRCC-2 to −4) treated with preoperative axitinib, tumor tissues showed heterogeneous pattern. These findings indicate that some parts of the cancer would show a good response to axitinib but other parts would not. On the other hand, in the present patient with cT3aN1M0, renal cell cancer with papillary type 2 (pRCC2), surgically resected cancer tissues (T1 to T3) showed high expression of pAkt (Ser-473) and pAkt (Thr-308), as well as very low expression of pS6, indicating that the mTORC2-Akt signaling may be more important for molecular targeting than the mTORC1-S6 pathway in HLRCC-associated kidney cancer compared with clear cell RCC

Article Snippet: We performed Western blotting using a rabbit anti-human antibody targeting pAkt (Ser-473) (Cell Signaling Technology, Inc; PhosphoPlus Akt (Ser-473) Antibody Kit; # 9270, Danvers, MA), a rabbit anti-human antibody for pAkt (Thr-308) (Cell Signaling Technology, Inc; Phospho-Akt (Thr308) Antibody Kit; # 2965, Danvers, MA), a rabbit anti-human antibody targeting phosphorylated ribosomal protein S6 kinase (pS6) (2 F9, Cell Signaling Technology, Inc; # 4856), as described previously [ ].

Techniques: Western Blot, Marker, Expressing

Journal: Journal of leukocyte biology

Article Title: Mammalian Target of Rapamycin regulates a hyper-responsive state in pulmonary neutrophils late after burn injury.

doi: 10.1002/JLB.3AB0616-251RRR

Figure Lengend Snippet: Burn or sham mice treated with rapamycin (4mg/kg/day) underwent an intra-tracheal inoculation with PAK at 14 dpi. A) 48 hours following infection with 1 × 106 CFU of PAK, bacterial burden was determined in whole lung homogenate. B) Following infection with 5 × 106 CFU of PAK, mice were monitored for morbidity for 72h, and moribund or deceased animals were recorded. C) Blood from the submandibular vein was collected and NO levels were directly measured. D) Total number of neutrophils in the lung is determined as (%neutrophils as live cells x live cells counted at harvest). E) Cells from whole lung tissue were collected at 14 d post burn injury and stimulated with FBS in vitro with (Rapa) or without (FBS) Rapamycin for 30 minutes. After gating on neutrophils, we show intracellular pS6 staining in three representative replicates from three repeat experiments. FMO refers to the Fluorescence Minus One control, which is the fluorescence signal using every antibody apart from anti-pS6. E-F) pS6 staining of gated neutrophils were measured and used to calculate F) MFI of cellular pS6 and G) pS6+ percentage of gated neutrophils. Data shown are +/−SEM. *p<0.05, **p<0.01 and representative of three repeated experiments. Numbers of mice in representative figures; A, C) sham, n=4; burn, n=6; B) sham, n=8; burn, n=8; D) sham, n=6; burn, n=6; F,G) sham, n=5; burn, n=5.

Article Snippet: To quantify S6 phosphorylation (pS6), cells were surface stained as above and then intracellularly stained with anti-pS6 antibody (eBioscience; San Diego, CA).

Techniques: Infection, In Vitro, Staining, Fluorescence, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: A Novel Origin for Granulovacuolar Degeneration in Aging and Alzheimer’s Disease: Parallels to Stress Granules

doi: 10.1038/labinvest.2011.149

Figure Lengend Snippet: Hippocampus sections from control cases rarely display pS6 granular staining (A). In AD cases, however, many neurons have specific pS6 granules (B). Higher magnification reveals the granules range from about 0.1 μm to 2.0 μm in diameter (C). Quantification of the number of neurons in the entire hippocampal section containing granules in the CA1, CA2, CA3, and CA4 regions, finds AD cases (n=11) contain nearly 20 fold more pS6 containing neurons than age-matched controls (n=8) (D,*p<0.01). Scale bars=20 μm.

Article Snippet: Antibodies used in this study are rabbit antibody against phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (pS6) (Cell Signaling 91B2), sheep phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (Abcam), mouse monoclonal to total S6 protein (Cell Signaling 54D2), rabbit antibody to p54/RCK (Bethyl Laboratories A300-460), Alz-50 (gift of Peter Davies), PHF1 (gift of S. Greenberg), and AT8 (Pierce Endogen), monoclonal anti-8-hydroxyguanosine (8OHG, QED Biosciences), and anti-rRNA (Santa Cruz Biotechnology).

Techniques: Control, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: A Novel Origin for Granulovacuolar Degeneration in Aging and Alzheimer’s Disease: Parallels to Stress Granules

doi: 10.1038/labinvest.2011.149

Figure Lengend Snippet: Compared with an untreated section of AD hippocampus (A), dephosphorylation of an adjacent tissue section completely abolished all immunoreactivity for pS6 (B). Adsorption with pS6 peptide (D) also abolished all granular staining as seen on adjacent section (C). * denotes landmark vessels in A and B, and C and D. Scale bars=50 μm.

Article Snippet: Antibodies used in this study are rabbit antibody against phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (pS6) (Cell Signaling 91B2), sheep phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (Abcam), mouse monoclonal to total S6 protein (Cell Signaling 54D2), rabbit antibody to p54/RCK (Bethyl Laboratories A300-460), Alz-50 (gift of Peter Davies), PHF1 (gift of S. Greenberg), and AT8 (Pierce Endogen), monoclonal anti-8-hydroxyguanosine (8OHG, QED Biosciences), and anti-rRNA (Santa Cruz Biotechnology).

Techniques: De-Phosphorylation Assay, Adsorption, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: A Novel Origin for Granulovacuolar Degeneration in Aging and Alzheimer’s Disease: Parallels to Stress Granules

doi: 10.1038/labinvest.2011.149

Figure Lengend Snippet: The granular staining can also be detected using a mouse monoclonal phosphorylation-independent S6 antibody, recognizing total S6 protein (E). In addition to the morphologically similar GVD structures (arrow), many pyramidal neurons in the AD cases demonstrated diffuse cytoplasmic reactivity. Double label fluorescent microscopy revealed many of the same structures are recognized by the antibodies to both pS6 (A) and total S6 (B) and (C, merged image). A polyclonal antibody to pS6 raised in sheep also specifically labels the GVD/stress granule structures in the AD cases (D, arrows). p54/RCK was also found to localize strikingly to the same structures (Figure F). Scale bars=25 μm.

Article Snippet: Antibodies used in this study are rabbit antibody against phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (pS6) (Cell Signaling 91B2), sheep phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (Abcam), mouse monoclonal to total S6 protein (Cell Signaling 54D2), rabbit antibody to p54/RCK (Bethyl Laboratories A300-460), Alz-50 (gift of Peter Davies), PHF1 (gift of S. Greenberg), and AT8 (Pierce Endogen), monoclonal anti-8-hydroxyguanosine (8OHG, QED Biosciences), and anti-rRNA (Santa Cruz Biotechnology).

Techniques: Staining, Phospho-proteomics, Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: A Novel Origin for Granulovacuolar Degeneration in Aging and Alzheimer’s Disease: Parallels to Stress Granules

doi: 10.1038/labinvest.2011.149

Figure Lengend Snippet: Double staining of pS6 (brown) with either Alz-50 (blue, A) or PHF1 (blue, B) of AD hippocampus. In both A and B, arrows mark neurons with no colocalization while arrowheads indicate colocalization with tau. Quantification of the entire CA1, CA2, CA3, and CA4 regions from 4 AD cases reveals that tau is present in approximately 40% ± 17% of those cells with pS6 positive granules (C).

Article Snippet: Antibodies used in this study are rabbit antibody against phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (pS6) (Cell Signaling 91B2), sheep phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (Abcam), mouse monoclonal to total S6 protein (Cell Signaling 54D2), rabbit antibody to p54/RCK (Bethyl Laboratories A300-460), Alz-50 (gift of Peter Davies), PHF1 (gift of S. Greenberg), and AT8 (Pierce Endogen), monoclonal anti-8-hydroxyguanosine (8OHG, QED Biosciences), and anti-rRNA (Santa Cruz Biotechnology).

Techniques: Double Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: A Novel Origin for Granulovacuolar Degeneration in Aging and Alzheimer’s Disease: Parallels to Stress Granules

doi: 10.1038/labinvest.2011.149

Figure Lengend Snippet: In AD hippocampal tissue sections, neurons with TUNEL-positive nuclei (blue, arrow) show no colocalization with pS6 granules (brown, arrowhead) (A). No colocalization of pS6 granules (brown, arrows) is observed with the bFGF assay which stains mostly extracellular NFT (blue) (B). Scale bars=50 μm.

Article Snippet: Antibodies used in this study are rabbit antibody against phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (pS6) (Cell Signaling 91B2), sheep phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (Abcam), mouse monoclonal to total S6 protein (Cell Signaling 54D2), rabbit antibody to p54/RCK (Bethyl Laboratories A300-460), Alz-50 (gift of Peter Davies), PHF1 (gift of S. Greenberg), and AT8 (Pierce Endogen), monoclonal anti-8-hydroxyguanosine (8OHG, QED Biosciences), and anti-rRNA (Santa Cruz Biotechnology).

Techniques: TUNEL Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: A Novel Origin for Granulovacuolar Degeneration in Aging and Alzheimer’s Disease: Parallels to Stress Granules

doi: 10.1038/labinvest.2011.149

Figure Lengend Snippet: Adjacent serial sections of AD hippocampus stained with pS6 (A) and rRNA (B). Following quantification of the cytoplasmic levels of rRNA, cells with pS6-positive granules (red circles) contain significantly less rRNA (*p<0.01).

Article Snippet: Antibodies used in this study are rabbit antibody against phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (pS6) (Cell Signaling 91B2), sheep phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (Abcam), mouse monoclonal to total S6 protein (Cell Signaling 54D2), rabbit antibody to p54/RCK (Bethyl Laboratories A300-460), Alz-50 (gift of Peter Davies), PHF1 (gift of S. Greenberg), and AT8 (Pierce Endogen), monoclonal anti-8-hydroxyguanosine (8OHG, QED Biosciences), and anti-rRNA (Santa Cruz Biotechnology).

Techniques: Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: A Novel Origin for Granulovacuolar Degeneration in Aging and Alzheimer’s Disease: Parallels to Stress Granules

doi: 10.1038/labinvest.2011.149

Figure Lengend Snippet: Adjacent serial sections of AD hippocampus stained for pS6 (A) and 8OHG (B). The majority of cells with pS6-positive granules contain visibly less 8OHG (red circles), while approximately 25% of the pS6-positive neurons have 8OHG levels comparable to cells not containing pS6 (black circles) Following quantification of the cytoplasmic levels of 8OHG, cells with pS6-positive granules contain significantly less 8OHG (C, *p<0.01).

Article Snippet: Antibodies used in this study are rabbit antibody against phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (pS6) (Cell Signaling 91B2), sheep phospho-pS6 ribosomal protein phosphorylated at Ser 235/236 (Abcam), mouse monoclonal to total S6 protein (Cell Signaling 54D2), rabbit antibody to p54/RCK (Bethyl Laboratories A300-460), Alz-50 (gift of Peter Davies), PHF1 (gift of S. Greenberg), and AT8 (Pierce Endogen), monoclonal anti-8-hydroxyguanosine (8OHG, QED Biosciences), and anti-rRNA (Santa Cruz Biotechnology).

Techniques: Staining

Journal: Nature communications

Article Title: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage.

doi: 10.1038/ncomms2008

Figure Lengend Snippet: Figure 1 | FOXO3 interacts with the ATM–Chk2–p53–H2AX complex. (a) Whole-cell lysates of MCF-7 treated with CPT (1 µM) for 2 or 4 h or DMSO (0, control) were subjected to immunoprecipitation (IP) with an anti-FOXO3 antibody (Ab) or an isotype IgG (control) followed by immunoblotting (IB) with Abs as indicated, or an anti-FOXO3 (IP control) or a control Ab against IgG light chain (L). (b–h) Similarly, cell lysates were subjected to IP with an Ab against ATM-pS1981 (b), p53-pS15 (c),p53-pS20 (d), p53-pS46 (e),Chk2 (f), γ-H2AX (g), HIPK2 (h) or an isotype IgG followed by IB with the indicated Abs or a control Ab against IgG(L) or heavy chain (H).

Article Snippet: Antibodies against phospho-ATM Serine-1981 (ATM-pS1981, 1:1,000 dilution), phospho-Chk2 (Chk2-pT68, 1:1,000 dilution), p53 (1:1,000 dilution), phospho-p53 (p53-pS6, p53-pS9, p53-pS15, p53-pS20, p53pS37, and p53-pS46, 1:1,000 dilution), Bax (1:500 dilution) and Puma (1:500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Immunoprecipitation, Western Blot

Journal: Nature communications

Article Title: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage.

doi: 10.1038/ncomms2008

Figure Lengend Snippet: Figure 2 | FOXO3 is essential for the formation of ATM–Chk2–p53–H2AX nuclear foci. (a) Accumulation of a fraction of FOXO3 at laser-induced damage was detected in MCF-7 cells 30 min after focused laser micro-irradiation. The cells were stained with antibodies (Abs) against FOXO3 and γ-H2AX or ATM-pS1981, followed by fluorescence microscopy as described in the section of Immunofluorescence under ‘Methods’. DAPI was used to show the nuclei, and co-localizations of FOXO3 with ATM-pS1981 were shown as the merged images. Scale bar, 6 µm. (b–f) MCF-7 cells were treated with CPT (1 µM) or DMSO control for 2 h, and co-localizations between FOXO3 and p53-pS15 (b), p53-pS20 (c), p53-pS46 (d), γ-H2AX (e) or ATM- pS1981 (f) were detected using Abs as indicated and followed by fluorescence microscopy. Scale bar, 6 µm. (g–l) FOXO3 is necessary for the formation of p53-pS15, p53-pS20 and p53-pS46, γ-H2AX, ATM-pS1981 and Chk2-pT68 nuclear foci on CPT-induced DNA damage. MCF-7 stable cell lines transfected with FOXO3-shRNA or control-shRNA were treated with CPT (1 µM) or DMSO for 2 h, and then the sub-cellular localizations and co- localization of FOXO3 and p53-pS15 (g), p53-pS20 (h), p53-pS46 (i), γ-H2AX (j), ATM-pS1981 (k) or Chk2-pT68 (l) were detected using specific Abs as indicated. An average of 200 cells with specific nuclear foci in each comparison was determined and shown. The images of nuclear foci of these proteins were shown in Supplementary Fig. S5. The error bars represent standard deviation, and the statistical test is the paired t-test.

Article Snippet: Antibodies against phospho-ATM Serine-1981 (ATM-pS1981, 1:1,000 dilution), phospho-Chk2 (Chk2-pT68, 1:1,000 dilution), p53 (1:1,000 dilution), phospho-p53 (p53-pS6, p53-pS9, p53-pS15, p53-pS20, p53pS37, and p53-pS46, 1:1,000 dilution), Bax (1:500 dilution) and Puma (1:500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Irradiation, Staining, Fluorescence, Microscopy, Immunofluorescence, Control, Stable Transfection, Transfection, shRNA, Comparison, Standard Deviation

Journal: Nature communications

Article Title: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage.

doi: 10.1038/ncomms2008

Figure Lengend Snippet: Figure 3 | FOXO3 is required for ATM–Chk2–p53 phosphorylation and PARP-1 cleavage. (a) MCF-7 stable cell lines transfected with FOXO3-shRNA or control-shRNA were treated with CPT (1 µM) or DMSO (negative control) for 4 h, and whole-cell lysates were prepared and the levels of proteins were analysed by IB with specific antibodies (Abs) as highlighted or an anti-β-actin (loading control). (b,c) A549 (b) and H1299 (c) stable cell lines transfected with FOXO3-shRNA or control-shRNA were treated with CPT (1 µM) or DMSO (negative control) for 4 h, and whole-cell lysates were analysed by IB with specific Abs as indicated or an anti-β-actin. (d) Cell lysates from MCF-7 stable cell lines (FOXO3-shRNA and control-shRNA) treated with CPT (1 µM) for an indicated time course were subjected to IB analysis using Abs against PARP-1 and β-actin (loading control).

Article Snippet: Antibodies against phospho-ATM Serine-1981 (ATM-pS1981, 1:1,000 dilution), phospho-Chk2 (Chk2-pT68, 1:1,000 dilution), p53 (1:1,000 dilution), phospho-p53 (p53-pS6, p53-pS9, p53-pS15, p53-pS20, p53pS37, and p53-pS46, 1:1,000 dilution), Bax (1:500 dilution) and Puma (1:500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Stable Transfection, Transfection, shRNA, Control, Negative Control

Journal: Nature communications

Article Title: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage.

doi: 10.1038/ncomms2008

Figure Lengend Snippet: Figure 5 | Phosphorylated ATM–Chk2–p53 may be needed for FOXO3-mediated apoptosis. (a) MCF-7 cells were transfected with control-siRNA (control) or siRNA (0.3 µM) targeting ATM, Chk2 or p53 and incubated for 48 h, and then these cells were treated with CPT (1 µM) ( + ) or DMSO ( − ) for 48 h. Whole-cell lysates were prepared from these treated cells and the levels of PARP-1 and its degraded proteins, ATM, Chk2, p53 and their phosphorylated proteins or a p53 target (Bax) were analysed by IB with specific antibodies (Abs) as indicated or an anti-β-actin (loading control). (b) MCF-7 cells were first treated with ATM inhibitor (Mirin) or Chk2 inhibitor (NSC10955) or p53 inhibitor (Pifithrin), 20 µM of each, or DMSO for 6 h, and then treated with CPT (1 µM) or DMSO for 16 h. Whole-cell lysates were prepared from these treated cells and the levels of PARP-1 and its degraded proteins, and the indicated proteins and β-actin were analysed by IB analysis with specific Abs. (c) MCF-7 cells were transfected with p53-siRNA or Cont-siRNA (Control-siRNA) for 48 h. Whole-cell lysates were prepared from these transfected cells and the expressions of p53 and FOXO3 (control) were determined by IB analysis with specific Abs. (d) MCF-7 (p53-siRNA) cells were transfected with pcDNA3 (control) or p53 wild-type (WT) or mutant (p53-S15A or p53-S20A or p53-46A) expression vectors (2 µg DNA for each) for 48 h, and then treated with CPT (1 µM) ( + ) or DMSO ( − ) for 48 h. Whole-cell lysates were prepared from these treated cells and the levels of PARP-1 and its degraded proteins, and the indicated proteins were analysed by IB analysis with specific Abs. (e) MCF-7 (p53-siRNA) cells were transfected with control and expression vectors for 24 h, the transfected cells were treated with CPT (1 µM) or DMSO control for 36 h, and cellular apoptosis was determined by TUNEL assays (Promega). Images of apoptotic cells were shown in Supplementary Fig. S9. An average (%) of TUNEL-positive (apoptotic) cells was determined and shown in the diagram. **P = 0.0001 (paired t-test).

Article Snippet: Antibodies against phospho-ATM Serine-1981 (ATM-pS1981, 1:1,000 dilution), phospho-Chk2 (Chk2-pT68, 1:1,000 dilution), p53 (1:1,000 dilution), phospho-p53 (p53-pS6, p53-pS9, p53-pS15, p53-pS20, p53pS37, and p53-pS46, 1:1,000 dilution), Bax (1:500 dilution) and Puma (1:500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Control, Incubation, Mutagenesis, Expressing, TUNEL Assay

Journal: Nature communications

Article Title: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage.

doi: 10.1038/ncomms2008

Figure Lengend Snippet: Figure 6 | FOXO3 has a role in regulating chromatin retention of phosphorylated p53. (a) MCF-7 stable cell lines transfected with FOXO3-shRNA or control-shRNA were treated with CPT (1 µM) ( + ) or DMSO ( − ) (control) for 4 h, and cells were collected and fractionated with Nonidet P-40 as described in the section of Chromatin Retention Assay under ‘Methods’. Equal amount (20 µg) of each fraction was analysed by IB analysis with the highlighted antibodies as described above. Proteins β-tubulin, lamin A/C and HMG14 (high-mobility-group 14, a chromosome-binding protein) represent the fractionation and loading controls of the cytosol (fraction I), the nucleoplasm (fraction III) and the chromatin (fraction IV), respectively. (b–f) MCF-7 (control-shRNA) and MCF-7 (FOXO3-shRNA) cells were transfected with p53 siRNA for 48 h, then transfected with pcDNA3 (negative control) (b) or the p53-WT vector (c) or the specific vectors expressing p53-S15D (d), p53-S20D (e) and p53-S46D (f) for 36 h. The transfected cells were treated with CPT (1 µM) or DMSO for 4 h, then cells were collected and subjected to chromatin fractionation and IB analysis as described above.

Article Snippet: Antibodies against phospho-ATM Serine-1981 (ATM-pS1981, 1:1,000 dilution), phospho-Chk2 (Chk2-pT68, 1:1,000 dilution), p53 (1:1,000 dilution), phospho-p53 (p53-pS6, p53-pS9, p53-pS15, p53-pS20, p53pS37, and p53-pS46, 1:1,000 dilution), Bax (1:500 dilution) and Puma (1:500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Stable Transfection, Transfection, shRNA, Control, Binding Assay, Fractionation, Negative Control, Plasmid Preparation, Expressing

Journal: Nature communications

Article Title: FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage.

doi: 10.1038/ncomms2008

Figure Lengend Snippet: Figure 7 | A link between FOXO3 and the ATM–Chk2–p53-mediated apoptotic programme. A schematic shows the FOXO3-dependent activation of ATM-pS1981, Chk2-pT68, HIPK2, p53-pS15, p53-pS20, p53-pS46 and the possible roles of p53 and Hdm2 in downregulation of FOXO3 as negative feedback loops, and the downstream p53 apoptotic- signalling pathway after DNA damage induced by CPT. The dashed lines denote the inhibitory pathways according to the published literature.

Article Snippet: Antibodies against phospho-ATM Serine-1981 (ATM-pS1981, 1:1,000 dilution), phospho-Chk2 (Chk2-pT68, 1:1,000 dilution), p53 (1:1,000 dilution), phospho-p53 (p53-pS6, p53-pS9, p53-pS15, p53-pS20, p53pS37, and p53-pS46, 1:1,000 dilution), Bax (1:500 dilution) and Puma (1:500 dilution) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay